KDO Aldolase and condensation reactions employed therewith

Abstract

Aureobacterium barkerei strain KDO-37-2 (ATCC 49977) KDO aldolase (EC 4.1.2.23) isolated therefrom are disclosed. The DKDO aldolase is further disclosed to have a broad substrate specificity with respect to its reverse reaction, i.e. the condensation of aldoses with pyruvate to form a wide range of 2-keto-3-deoxy-onic acids, including 2-keto-3-deoxy-nonulosonic acid, 2-keto-3-deoxy-octulosonic acid, 2-keto-3-deoxy-heptulosonic acid, and 2-keto-3-deoxy-hexulosonic acid. In particular, 3-deoxy-D-manno-2-octulosonic acid (D-KDO), a vital component of lipopolysaccharides found in the bacterial outer membrane may be synthesized from D-arabinose and pyruvate in 67% yield. Additionally, protected forms of the KDO aldolase products, e.g. hexaacetyl 2-keto-3-deoxy-nonulosonic acid and pentaacetyl 2-keto-3-deoxy-octulosonic acid, may be decarboxylated to form the corresponding 2-deoxy-aldoses, e.g. 2-deoxy-octulose and 2-deoxy-heptulose respectively.

What is claimed is: 
     
       1. A method for synthesizing 2-keto-3-deoxy-onic acids and analogs thereof, the 2-keto-3-deoxy-onic acids being selected from the group consisting of 2-keto-3-deoxy-hexulosonic acid, 2-keto-3-deoxy-heptulosonic acid, 2-keto-3-deoxy-octulosonic acid, and 2-keto-3-deoxy-nonulosonic acid, the 2-keto-3-deoxy-octulosonic acid excluding 3-deoxy-D-manno-2-octulsonic acid, the method comprising the following steps, viz.: Step A: providing a purified KDO aldolase; then   Step B: admixing, in an aqueous solution, a 3-6 carbon aldose, exclusive of D-arabinose, an excess of pyruvate, and a catalytic amount of the purified KDO aldolase from said Step A; and then   Step C: incubating the admixture of said Step B for condensing the 3-6 carbon aldose with the pyruvate for producing the 2-keto-3-deoxy-onic acid.   
     
     
       2. A method for synthesizing a 2-keto-3-deoxy-onic acid as described in claim 1, wherein: in said Step B, the 3-6 carbon aldose having an R-configuration at position C-3.   
     
     
       3. A method for synthesizing 3-deoxy-α-D-altro-2-octulosonic acid as described in claim 1, wherein: in said Step B, the 3-6 carbon aldose is D-ribose.   
     
     
       4. A method for synthesizing 3,5-dideoxy-α-D-manno-2-octulosonic acid as described in claim 1, wherein: in said Step B, the 3-6 carbon aldose is deoxy-D-ribose.   
     
     
       5. A method for synthesizing 3-deoxy-α-D-arabino-2-heptulosonic acid as described in claim 1, wherein: in said Step B, the 3-6 carbon aldose is D-erythrose.   
     
     
       6. A method for synthesizing 3-deoxy-α-D-threo-2-hexulosonic acid as described in claim 1, wherein: in said Step B, the 3-6 carbon aldose is D-glyceraldehyde.   
     
     
       7. A method for synthesizing 3-deoxy-α-D-lyxo-2-heptolosonic acid as described in claim 1, wherein: in said Step B, the 3-6 carbon aldose is D-threose.   
     
     
       8. A method for synthesizing 3-deoxy-α-L-erythro-2-hexulosonic acid as described in claim 1, wherein: in said Step B, the 3-6 carbon aldose is L-glyceraldehyde.   
     
     
       9. A method for synthesizing 2-keto-3-deoxy-onic acids and analogs thereof as described in claim 1, wherein: in said Step A: the purified KDO aldolase being isolated from Aureobacterium barkerei strain KDO-37-2.   
     
     
       10. A method for synthesizing 2-keto-3-deoxy-onic acids and analogs thereof as described in claim 1, wherein: in said Step B, the 3-6 carbon aldose being selected from the group consisting of D-threose, D-erythrose, D-ribose, 2-deoxy-D-ribose, L-glyceraldehyde, D-glyceraldehyde, 2-deoxy-2-fluoro-D-arabinose, D-lyxose, 5-azido-2,5-dideoxy-D-ribose, D-altrose, and L-mannose.   
     
     
       11. A method for synthesizing 3-deoxy-D-manno-2-octulsonic acid and analogs thereof comprising the following steps, viz.: Step A: providing a purified KDO aldolase isolated from Aureobacterium barkerei strain KDO-37-2; then   Step B: admixing, in an aqueous solution, a catalytic amount of the purified KDO aldolase from said Step A with D-arabinose or an analog thereof and an excess of pyruvate; then   Step C: incubating the admixture of said Step B for condensing the D-arabinose or its analog with the pyruvate for producing the 3-deoxy-D-manno-2-octulsonic acid with a yield of greater than 50%.